1. Field Of The Invention
This invention relates to a method for speciating and identifying paraffinophilic materials.
2. Description Of The Prior Art
Human immunodeficiency virus type 1 or HIV causes acquired immunodeficiency syndrome ("AIDS") which is a fatal disease approaching epidemic proportions throughout the world. By current estimates, 110 million people will be infected with HIV by the year 2010. When AIDS develops, it is usually characterized by opportunistic infection, such as Pneumocystic pneumonia, Kaposi's sarcoma, lymphoma and Mycobacterium avium intracellulare complex (MAI).
It has been found that more than 80% of AIDS patients have MAI present in their bodies (J. M. Wallace et al., Mycobacterium avium complex in patients with AIDS--A Clinicopathologic Study. Chest 93 (5) 926-932 (1988)). Organisms of MAI prior to the AIDS epidemic were recognized as a rare form of pneumonia in patients with chronic lung infections (E. Wolinsky, Nontuberculous mycobacteria and associated diseases. Am. Rev. Respir. Dis. 1979, 119:107-59). Organisms of MAI comprise two closely related species, M. avium and M. intracellulare, which have minor virulence in the non-HIV host. By 1980, only 24 cases of MAI had been reported in the medical literature (C. R. Horsburgh, Jr. et al., Disseminated infection with Mycobacterium avium intracellulare. Medicine (Baltimore) 1985, 64:36-48). However, the epidemic of disseminated MAI infection is concurrent with the AIDS epidemic.
Studies have shown that disseminated MAI infection makes a substantial contribution to both morbidity and mortality in AIDS patients (C. R. Horsburgh, Jr. et al., The Epidemiology of Disseminated Nontuberculous Mycobacterial Infection in the Acquired immunodeficiency syndrome (AIDS). Am. Rev. Respir. Dis. 1989, 139:4-7).
Up to the present time, the most common source of isolating MAI clinically has been by means of the blood. Isolation techniques for determining the presence or absence of MAI in the patients'blood are known. One method involves using the BACTEC Radiometric System, which is a product of the Johnson Division of Becton and Dickenson. The system itself utilizes hemoculture tubes that contain Middlebrook 7H12 liquid broth plus 0.05% (v/v) sodium polyanethyl sulphonate in hemoculture vials. In addition, the 7H12 broth contains Carbon-14 labelled palmitic acid. In use, vials containing mycobacterial growth give off Carbon-14 labelled CO.sub.2 and this is detected by a device similar to that used for liquid scintillation capable of detecting beta emitters. Another method of isolation in blood involves using genetic probes which rely upon DNA hybridization (C. M. Reichert et al., Pathologic features of AIDS. In: V. T. DeVita Jr. et al. (eds) AIDS etiology, diagnosis, treatment and prevention, p. 134 NY J. 13, Lipponcott, 1985).
However, when MAI becomes widely disseminated in AIDS patients the involvement by way of the blood of bone, lungs, spleen and the CNS causes an almost 100% rate of mortality (C. C. Hawkins et al., MAI in patients with acquired immunodeficiency syndrome. Am. Intern. Med. 1986; 105: 184-8) (J. Hoy et al., Quadruple drug therapy for Mycobacterium avium intracellulare bacteremia in AIDS patients. J. Infect. Dis. 990; 161: 801-5).
These known methods, although effective, require expensive equipment and specialized operating personnel and materials. Thus, smaller hospital centers where few AIDS patients are seen, field laboratories, and third world countries, where resources are limited, do not have this specialized equipment and personnel. A simpler and more inexpensive method and apparatus of isolating and identifying MAI would be of substantial benefit in such situations.
It is known that many atypical Mycobacteria grow on basal salt media devoid of any carbon sources other than paraffin wax which is introduced into the media in the form of paraffin was coated roads. Fuhs, G. W., "Der Mikrobiell Abbau Von Kohlenwasserstoffen", Arch. Mikrobiol. 39:374-422 (1961). Mishra, S. K. et al., "Observations On Paraffin Baiting As a Laboratory Diagnostic Procedure in Nocardiosis", Mycopathologica and Mycologia Applicata 51 (2-3): 147-157 (1973) utilized paraffin coated rods and basal salt medium to isolate Nocardia asteroides from clinical specimens such as sputum, bronchial lavage and cerebrospinal fluid.
The technique was further improved by substituting paraffin wax coated slides for rods and thereby making possible the use on an in situ Kinyoun cold acid-fastness staining procedure for organisms growing on the paraffin coated slide. Ollar, R. A., "A Paraffin Baiting Technique that Enables a Direct Microscopic View of in situ Morphology of Nocardia asteroides with the Acid-Fast or Fluorescence Staining Procedures", Zbl. Bakt. Hyg., Abt. Orig. A, 234: 81-90 (1976). With this assay, a positive reaction tells the user immediately that a mycobacteria organism other than M. tuberculosis is present.
U.S. Pat. No. 5,153,119, which names one of the joint inventors of the present invention as sole inventor, discloses a method for speciating and identifying MAI in a specimen and involves the use of paraffin coated slides to determine the presence or absence of atypical Mycobacteria (mycobacteria other than M. tuberculosis, M. laprae, and M. paratuberculosis). This process, while quite effective for isolating and speciating of MAI, may for some purposes be deemed to be relatively slow, taking on the order of about 6 days (in feces) to 34.5 days (in blood). The disclosure of this patent is expressly incorporated herein by reference.
As will be apparent from the foregoing, it is vital to human health that MAI be identified and treated as early as possible (C. A. Kemper et al., California Collaborative Group; Microbiologic and clinical response of patients with AIDS and MAC bacteremia to a four oral drug regimen; In: Program and abstracts of the 30th Interscience Conference on Antimicrobial Agents and Chemotherapy; Atlanta, Oct. 21-24, 1990; Washington D.C.; Am. Society for Microbiology; 1990; 297. abstract).
There is a real and substantial need for improved means of rapidly determining the presence of MAI and other paraffinophilic organisms in a patient.